Compaction of replicated chromosomes into morphologically and spatially distinct sister chromatids is essential for faithful DNA segregation in all organisms. This is a complex, poorly understood process that is controlled by the physical and mechanical properties of DNA as well as the action of specific proteins and enzymes. In bacteria, only a small set of essential factors are required for chromosome compaction, offering the hope that it will be relatively straightforward to understand the principles by which they act. We have developed and adapted a new set of molecular and cytological assays that will allow us to address specific steps in DNA compaction and organization in the model organism Bacillus subtilis. Here, newly replicated DNA is compacted by the combined activities of topoisomerases that generate interwound (supercoiled) loops, small nucleoid-associated proteins that bend DNA, and SMC condensin complexes, which are thought to bridge DNA segments. We know that the organization of the compacted chromosome involves folding both at short and long length-scales, but what these two levels of organization look like and how they are coordinated are unclear. Using genome-wide chromosome conformation capture complemented by quantitative cytological assay, we will define compaction on short length-scales and chromosome organization on long length-scales. This new level of description will then allow us to establish the contributions of and interplay between the small set of essential factors that compact the chromosome and the transcription machinery that opens up the DNA. In particular, we aim to gain insight into the role of the highly conserved SMC condensin complex, for which we have the least information. Our preliminary data indicate that important factors that participate in chromosome compaction and segregation have been missed by traditional genetic screens, and we will take a novel cytological approach and high throughput synthetic lethal screens to identify them. Our specific aims are to: 1) Determine how the SMC condensin complex contributes to chromosome conformation in vivo; define the role of ParB/parS and highly transcribed genes in SMC-mediated origin segregation; and establish how SMC is enriched at these loci. 2) Define how DNA is compacted on short length-scales throughout the chromosome using genome-wide interaction frequencies. 3) Identify and characterize new chromosome organization and segregation factors using high throughput cytological and synthetic lethal screens.